IGF-I PROMOTES SCHWANN CELL MOTILITY VIA PHOSPHATIDYLINOSITOL-3 KINASE AND RAC GTPase.
Cheng H-L, Steinway M., Xin X., Feldman E. Department of Neurology, University of Michigan, Ann Arbor, MI 48109.
Schwann cell (SC) motility is important for myelin sheath formation
and nerve regeneration. We have previously reported that IGF-I
mediates Schwann cell (SC) process extension, focal adhesion kinase
(FAK) phosphorylation, increases cell motility and SC attachment
to axons. In the current study, we examined the role of phosphatidylinositol-3
(PI-3) kinase signaling in these phenomena. SC were cultured on
coverslips and serum deprived for 6 h before IGF-I treatment.
30 min of 10 nM IGF-I treatment induced cell process extension
and cytoskeletal rearrangement, as demonstrated by fluorescein
phalloidin staining. Addition of 10 mM
LY294002 (a PI-3 kinase inhibitor) but not PD98059 (a MEK inhibitor)
blocked the effects of IGF-I on cell process extension, cytoskeletal
rearrangement and SC attachment to axons. We next examined the
roles of PI-3 kinase and rac in IGF-I induced cell motility. Cell
motility was measured using a gold particle motility assay. 2
x 10(exp4) SC were seeded on BSA coated coverslips covered with
colloidal gold particles in serum free media +/- IGF-I. Cell motility
was determined by areas of gold particle free tracts behind the
moving cells. IGF-I enhances SC motility in a dose-dependent manner
which was blocked by 30 min pretreatment with 10 mM
LY294002. SC transfected with consecutively active (V12) rac are
more motile than control cells. In dominant negative (N17) rac
transfected cells, IGF-I enhancement of SC motility was diminished.
LY294002 has no effect on constitutively active rac mediated SC
motility, suggesting rac is downstream of PI-3 kinase. We also
examined the effects of LY294002 and rac mutants on IGF-I induction
of FAK phosphorylation. Se-rum-deprived SC were treated with 10
nM IGF-I +/- 10 mM LY294002 for 5-30
min. Whole cell lysates were collected and FAK immunoprecipitation
was performed followed by phosphotyrosine immunoblots. LY294002
blocks IGF-I induced FAK phosphorylation. V12 rac transfection
enhances FAK phosphorylation, and N17 rac blocks IGF-I effects
on FAK phosphorylation, suggesting FAK is downstream of rac. Our
data imply the PI-3 kinase pathway (PI-3 kinase-rac-FAK) is responsible
for IGF-I induced cell motility and FAK phosphorylation. Supported
by NIH RO1 NS32843 (ELF), ROI NS38849 (ELF) and grants from the
American Diabetes Association and Juvenile Diabetes Foundation
(ELF).
SCHWANN CELLS PROMOTE AXONAL OUTGROWTH IN hNT2 CULTURES
De Jonge R.R., Van Schaik I.N., Vermeulen M., Brand A., Kwa M.S.G., Baas F. Neurozintuigen Laboratorium, Academical Medical Center, Amsterdam, The Netherlands.
There is increasing evidence both clinically and laboratory supported that the axon may well play a pivotal role in the pathogenesis of demyelinating neuropathies, like the hereditary motor and sensory neuropathies and the acute and chronic inflammatory demyelinating polyneuropathies. Schwann cell-axon interactions may explain the clinical course and outcome in these patients. Until now it has not been possible to study these interactions in vitro using human Schwann cells and human neurons. The initial goal was to identify the proper culture conditions for these studies. Human Schwann cells obtained from nerve biopsies and amputation material and NTera 2/cl.D1 (hNT2) cells, a human teratocarcinoma cell line, were cultured as described previously. Retinoic acid was used to differentiate the hNT2 cells. First, the effect of different tropic factors (1-methyl-3isobutylxanthine, heregulin, forskolin and insulin) added to the culture media was studied. 1-methyl-3-isobutylxanthine and forskolin, factors that increase cAMP-levels, and Schwann cell conditioned medium showed promotion of the axonal outgrowth. Secondly, co-cultures were performed. These experiments showed I) the survival of differentiated hNT2 cells was prolonged and II) the differentiation of hNT cells was promoted in the presence of Schwann cells.
INFLAMMATORY INFILTRATION IN SURAL NERVE BIOPSIES OF PATIENTS WITH POLYNEUROPATHY ASSOCIATED WITH MONOCLONAL GAMMOPATHY
Eurelings M., Notermans N.C., van den Berg L.H., Jansen G.H., Bosboom W.M.J., Wokke J.H.J. University Hospital of Utrecht, the Netherlands
Tcells are considered to play a role in the pathogenesis of CIDP as Tcell infiltration is present in sural nerves, while polyneuropathy associated with monoclonal gammopathy may be mediated by antibody activity to the myelin associated glycoprotein (MAG). In this study we investigated whether the number of Tcells differentiates between polyneuropathy associated with M-protein with and without anti-MAG activity. The numbers of Tcells in sural nerve biopsies of 28 patients with polyneuropathy associated with IgM, 14 patients with IgM with anti-MAG activity, and 18 patients with IgG were compared with autopsy controls (5), chronic idiopathic axonal polyneuropathy (CIAP) (15) and CIDP (24). The number of Tcells was correlated with the clinical and electrophysiological features of these patients. The number of Tcells in polyneuropathy associated with IgM with anti-MAG activity (median 11; range 5-57 Tcells/mm2) was lower than in patients with IgM without anti-MAG activity and IgG, (p=0.03), and in the same range as in CIAP and autopsy controls (9; 3-21 Tcells/mm2). The number of Tcells in patients with IgM without anti-MAG activity and IgG (19; 1-75 Tcells/mm2) was in the same range as in CIDP (16; 6-113 Tcells/mm2). The number of Tcells in demyelinating polyneuropathy without anti-MAG activity (26; 5-57 TceIls/mm2) was higher than in axonal polyneuropathy (14; 1-75 Tcells/mm2). This results suggest that polyneuropathy associated with an IgM M-protein with anti-MAG activity is not Tcell mediated. Patients with a demyelinating polyneuropathy associated with an IgG or an IgM M-protein without anti-MAG activity and with a high number of Tcells may have CIDP.
DISTRIBUTION OF GALNAC-GD1A IN HUMAN NERVOUS TISSUES
Matsumoto A.(1), Yoshino H.(1), Yamada M.(2) (1)Dept. of Neurology, Kohnodai Hospital, National Center of Neurology & Psychiatry, Chiba , (2)Dept. of Pathology, Niigata University, Niigata, Japan
There have been reports suggesting that anti ganglioside antibodies are implicated in the pathogenesis of some neurological disorders, such as Guillain-Barré syndrome (GBS) or some cases with motor neuron disease (MND). Anti GalNAc-GDla antibody has been reported to exist in cases with GBS without sensory disturbance and axonal form of GBS. However, the pathological role of Ga1NAc-GDla antibody in developing GBS or MND has yet to be elucidated. Therefore, we have studied the distribution of GalNAc-GDla in human nervous tissues using immunohistochemical technique. We also screened serum of neuropathies and MND, and confirmed the presence of anti GalNAc-GD1 a antibody in some cases with MND. Immunohistochemical study revealed that the axon of human anterior spinal root and anterior horn cells were stained with GalNAc-GDla antibody, but the posterior root was not. This result corresponded with the biochemical analysis by TLC immunostaining that showed GalNAc-GDla was detected only on the anterior root, but not on the posterior root. In conclusion, we have shown that GalNAc-GDla is exclusively expressed on the axon of motor nerve in human nervous tissues. The presence of anti GalNAc-GD1a antibody in some cases with GBS and MND considered, this specific localization of GalNAc-GDla, serving as a target antigen, might explain the selective involvement of motor nerve or motor neurons in the pathogenesis of motor neuropathy or MND.
PROMOTION OF AXONOPATHIES BY CERTAIN ESTERASE INHIBITORS: THE SEARCH FOR TBE MOLECULAR TARGET
Moretto A., Nicolli A., Lotti M. University of Padua, Padua, Italy
Organo-phosphates/phosphinates, sulfonyl fluorides, and (thio)carbamates exacerbate axon-opathies (promotion). This effect might be the result of interference with compensation/repair mechanism(s). Organophosphate induced delayed polyneuropathy (OPIDP) in hens is the model axonopathy. All promoters are inhibitors of neuropathy target esterase (NTE, the membrane-bound protein, target of OPIDP) which is defined as the phenyl valerate esterase (PVE) activity resistant to paraoxon and sensitive to mipafox (40 and 50 mM, respectively). However, promotion is not correlated with NTE inhibition although available data suggest that the target of promotion might be similar to it. A titration curve of paraoxon-resistant PVEs by mipafox dissected in nervous tissue homogenates another PVE, besides NTE (IC50 about 7 mM), with an IC50 of approx. 200 mM (M200). The promoters phenylmethanesulfonyl fluoride and butylsulfonyl fluoride had similar IC50s for both NTE and M200 (about 100 and 60 mM, respectively). NTE IC50s for the OPIDP initiators diisopropylfluoro phosphate (DFP) and dibutyldichlorovinyl phosphate were 0.2-0.5, 0.04-0.08 mM, respectively, whereas those for M200 were 2-10 and 0.06-0.2 mM, respectively. These results indicate that M200 is affected by promoters only. In vivo data confirmed the association between M200 inhibition and promotion: in fact, among others, the promoter KBR 2822 (0-(2-chloro-2,3-trifluoro-cyclobutyl) O-ethyl S-propyl ester) and DFP selectively inhibited M200 and NTE in hens, respectively. M200 activity is enriched in the soluble fraction of peripheral nerves. Interactions of M200 with a variety of inhibitors led to the identification of a protein which was partially purified with Sephacryl S-300 and Q-Sepharose columns and found to have a molecular weight of 50-60 kDa.
INTERACTION OF INTEGRIN AND CHEMOKINE IN THE PATHOGENESIS OF EAN IN LEWIS RATS. INHIBITION OF INTEGRINS WITH SYNTHETIC PEPTIDE RGD
Purev E., Kremlev S.G., Rostami A.M. University of Pennsylvania, Philadelphia, Pennsylvania, USA
This work was done to determine whether treatment with tripeptide Arg-Gly-Asp (RGD) can modify the expression of VLA-4 (a4bl integrin) and proinflammatory chemokines, MIP-1a and MCP-1, and the course of active experimental autoimmune neuritis (EAN). VLA-4 can mediate cell-cell and cell-extracellular matrix adhesion by binding to fibrinectin (FN) or vascular cell adhesion molecule 1 (VCAM-1). Blockade of either VLA-4 or VCAM-I with specific monoclonal antibodies significantly diminished leukocyte infiltration in sciatic nerve and attenuated the disease severity. RGD sequence is used as a receptor recognition motif for most integrin ligands. This study showed low levels of expression of VLA-4, MIP-la, and MCP-1 assessed by RT-CQ-PCR in naive rats; while in Lewis rats injected with SP-2653-78 aa, the levels increased and peaked at day 12-15 postimmunization (p.i.) and subsequently declined thereafter. The RGD-treated EAN rats had milder disease course, fewer infiltrating leukocytes and lower (P<0.05) levels of VLA-4, MIPla, and MCP-1 expression in the caudae equinae. In vitro, treatment of peritoneal macrophages elicited from normal and EAN animals with graded concentrations of MIP-1a and MCP-1 in the presence or absence of FN, SP-26 and RGD peptide, showed the same correlation. RGD peptide inhibited the expression of VLA-4, MIP-la or MCP-1, downregulated the clinical course of EAN and may be useful in suppression of inflammatory demyelination in human Guillain-Barré syndrome.
THE CELLULAR TRAFFICKING OF WILD TYPE AND MUTANT PMP22 PROTEINS IDENTIFIES SOME PATHOMECHANISMS IN THE PERIPHERAL NEUROPATHIES
Tobler A.L., Notterpek L., Naef R., Taylor V., Suter U., Shooter E.M. Dept. Neurobiology, Stanford Univ. School of Medicine, Stanford, CA, USA, Inst. Cell Biology, Swiss Federal Institute of Technology, Zurich, Switzerland
The peripheral myelin protein 22 (PMP22) plays a role in the formation, stability and maintenance of peripheral myelin. Duplications, deletions or mutations in the PMP22 gene underlie a set of inherited peripheral neuropathies. The heterozygous Trembler J (TrJ) mouse with a mutation (L16P) in PMP22 is a model for the severe form of CMT1A. The transport of an epitope-tagged TrJ-PMP22 is interrupted in the intermediate compartment (IC) of COS and Schwann cells preventing its insertion into the plasma membrane and disrupting the ER. Because TrJ-PMP22 forms a heterodimer with wt-PMP22 a fraction of the latter is also diverted to the IC when wt- and TrJ-PMP22 are coexpressed. The heterozygous TrJ Schwann cells are therefore stressed by a decrease in the transport of wtPMP22 to the membrane, by disruption of the ER and by intracellular accumulation of wt- and TrJ-PMP22 in the IC. In contrast when wt-PMP22 is overexpressed in Schwann cells to mimic the most common form of CMT1A the proteasome degradation pathway is overwhelmed and the ubiquitinated PMP22 accumulates in aggresomes. A more detailed comparison of these two different PMP22 trafficking pathways should produce a better understanding of how they lead to a similar disease phenotype.
IN VITRO SYSTEMS FOR THE STUDY OF HYPOXIC HYPOGLYCEMIC NEURONAL DEATH; ROLE OF PKCg
Sun X., Podratz J.L., Windebank A.J. Mayo Molecular Neuroscience Program, Rochester, MN 55905
Hypoxia due to small vessel disease and the metabolic effects of chronic hyperglycemia are thought to be major factors in producing peripheral nerve injury in diabetes. This may present acutely or subacutely in two forms, proximal asymmetric neuropathy and acute painful neuropathy accompanying weight loss or rigorous glycemic control. In both cases, the precipitant may be rapid normalization of blood sugar following a period of chronic hyperglycemia. We hypothesize that nerve cells that were protected from hypoxia by greater flux through glycolytic and anaerobic pathways die secondary to hypoxia when intracellular glucose rapidly falls. This has been difficult to test directly in animals. We have developed a model in which dorsal root ganglion neurons or PC12 cells were incubated in high glucose medium and low ambient oxygen. Acute lowering of glucose led to oxidative stress and neuronal apoptosis. This death was prevented by NGF in a dose dependent manner. Death was preceded by evidence of increased oxidative stress using dichlorodihydrofluorescein fluorescence. The increase in oxidative stress was not prevented by protective concentrations of NGF. Hypoxic, hypoglycemic neuronal death was also associated with significant elevation of PKCg. Inhibition of PKC, using sphingosine blocked NGF mediated neuroprotection. We propose that reciprocal reg-ulation of PKC by hyperglycemia and NGF is central to the neuronal injury induced by hypoxia and prevented by NGF. Since PKC inhibitors, antioxidants, and NGF are being used or proposed for clinical trials, it is critical to understand their potential mechanism of action in the nervous system.
WIDENINGS OF THE MYELIN LAMELLAE IN NEUROPATHIES ASSOCIATED WITH MONOCLONAL DYSGLOBULINEMIA: IgM, IgG or IgA. IMMUNOELECTRON MICROSCOPIC STUDY OF 5 CASES
Vallat J.M.(1), Sindou P.(1), Preux P.M.(1), Cros D.(2), Couratier P.(1) (1)Department of Neurology, University Hospital, Limoges 87042 FRANCE, (2)Department of Neurology, Massachusetts General Hospital, Boston 02114 MA USA
The lesions of peripheral nerve seen at the ultrastructural
level as widenings of myelin lamellae (WML) in patients with neuropathy
and IgM dysglobulinemia with anti-MAG are well documented. In
two such patients, we demonstrated by immunoelectron microscopic
examination (immunoEM) a colocalization of IgM and MAG in the
WML. In another patient, WML with IgM were noted, but we failed
to find any MAG in the WML; there was no serum anti-MAG or antiglycolipid
activity. WML have only rarely been found associated with monoclonal
IgG. No myelin antibodies were detected in the serum of our patient.
The IgG was visualized on immunoEM in the WML and in the form
of endoneurial deposits. There do not appear to be any descriptions
of WML associated with monoclonal IgA. In our case, both direct
and indirect immunofluorescence were strongly positive with IgA
and C3-d antibodies. WML were abundant involving most myelinated
fibers. IgA was detected by immunoEM in these WML. There was no
serum anti-MAG or antiglycolipid activity. High magnification
on EM evidenced differences in the appearance of the WML as a
function of the type of immunoglobulin. We concluded that in one
of our IgM cases and in our IgG and IgA cases, these immunoglobulins
were directed against, as yet unidentified antigens (not MAG).